The mammalian flavin-containing monooxygenases (FMOs) are enzymes distributed in the liver, lung and kidney of mammals. They catalyze the oxidation of a wide variety of foreign compounds or xenobiotics, including pesticides and narcotic drugs like nicotine. To date, five distinct members of this protein family, designated FMO 1, 2, 3, 4, and 5, have been identified in mammals including humans. Human FMO3, the dominant isoform of FMO in human liver, has been observed to exclusively convert nicotine to an all trans carcinogenic nicotine-N-1'- oxide. By contrast, the cytochrome P450s oxidize (S)-nicotine to a mixture of cis- and trans-N'-oxides. Interestingly, in smokers, the trans- N-1'-oxide ste has been proposed as a stereoselective functional biomarker for FMO3 in humans. Interestingly, the specific role of the FMOs relative to that of the cytochrome P450s in microsomal or in vivo oxidative metabolism of xenobiotics in mammals is not well defined, due to lack of non-substrate inhibitors specific and selective for the FMO isoforms. As a result, the relative contribution of FMOs in xenobiotic metabolism, is often deduced by inhibiting the reactions catalyzed primarily by the cytP450s. The lack of inhibitors selective for the FMOs may be due in part, to the paucity of information about the amino acid determinants of the substrate-binding sites of FMOs. The specific goal of this project therefore, is to identify the amino acid determinants of the substrate binding sites of especially, the long cancer implicated human FMO3 by: (1) using photoaffinity labeling studies followed by time-of- flight mass spectrometry (MALDI-TOF) to identify any covalently labeled residues. The probe to be used is photoactivatable N, N- dimethyl-4-azidoaniline, an azido-derivatized substrate analog of N, N- dimethylaniline, an FMO marker substrate. Oligonucleotide-mediated site-saturation mutagenesis will subsequently be used to ascertain whether residues, identified by the photolabeling/MALDI-TOF studies are critical for substrate binding. We hope the identification of residues crucial for substrate binding and catalysis will help us to search for and/or design FMO isoform selective inhibitors Availability of such inhibitors, should help researchers to do more definitive studies to determine the specific role of human FMO3 in the bioactivation of xenobiotics like nicotine to carcinogenic metabolism.